The invention relates to the causative agent of Mystery Swine Disease, the PRRS virus, to peptide sequences identified in the PRRS virus, and to incorporating these sequences in vaccines and diagnostic tests.
PRRS virus (PRRSV) is the causative agent of a pig disease, currently called porcine reproductive and respiratory syndrome (PRRS). The virus is the causative agent of a pig disease, seen since approximately 1987 in the U.S. and since 1990 in Europe, known initially under various names such as Mystery Swine Disease, Swine Infertility and Respiratory Syndrome, and many more. The virus itself was also given many names, among which Lelystad virus (LV), SIRS virus, and many more, but is now mostly designated porcine reproductive and respiratory syndrome virus (PRRSV). It causes abortions and respiratory distress in pigs and was first isolated in Europe in 1991 (EP patent 587780, U.S. Pat. No. 5,620,691) and subsequently in the U.S. and many other countries throughout the world. PRRSV is a small enveloped virus containing a positive strand RNA genome. PRRSV preferentially grows in macrophages. In addition to macrophages, PRRSV can grow in cell line CL2621 and other cell lines cloned from the monkey kidney cell line MA-104 (Benfield et al., J. Vet. Diagn. Invest. 4; 127-133, 1992). The genome of PRRSV, a polyadenylated RNA of approximately 15 kb was sequenced in 1993 (Meulenberg et al., Virology 192; 62-74, 1993). The nucleotide sequence, genome organization and replication strategy indicated that PRRSV is related to a group of small enveloped positive-strand RNA viruses, designated Arteriviruses. This group includes lactate dehydrogenase-elevating virus (LDV), equine arteritis virus (EAV), and simian hemorrhagic fever virus (SHFV). These viruses have a similar genome organization, replication strategy, morphology, and amino acid sequence of viral proteins. Arteriviruses contain a genome of 12.5 to 15 kb and synthesize a 3xe2x80x2 nested set of six subgenomic RNAs during replication. These subgenomic RNAs contain a leader sequence which is derived from the 5xe2x80x2 end of the viral genome. ORFs 1a and 1b comprise approximately two thirds of the viral genome and encode the RNA dependent RNA polymerase. Six smaller ORFs, ORFs 2 to 7, are located at the 3xe2x80x2 end of the viral genome. ORFs 2 to 6 likely encode envelope proteins whereas ORF7 encodes the nucleocapsid protein (Meulenberg et al, Virology 206; 155-163, 1995).
PRRSV is the first Arterivirus for which it has been demonstrated that all six proteins encoded by ORFs 2 to 7 are associated with the virion. The 15-kDa N protein (encoded by ORF7) and the 18-kDa integral membrane protein M (ORF6) are not N-glycosylated, whereas the 29- to 30-kDa GP2 protein (ORF2), the 45- to 50-kDa protein GP3 protein (ORF3), the 31-to 35-kDa GP4 protein (ORF4), and the 25-kDa protein GP5 (ORF5) are. These proteins have also been detected in extracellular virus and lysates of cells infected with a North American isolate of PRRSV, ATCC-VR2332, and other isolates of PRRSV (other isolates of PRRSV are for example CNCM I-1140, ECACC V93070108, CNCM I-1387, CNCM I-1388, ATCC-VR2402, ATCC-VR2429. ATCC-VR2430, ATCC-VR2431, ATCC-VR2475, ATCC-VR2385, but many others are known).
We earlier described the isolation and characterization of a panel of PRRSV-specific MAbs that were specific for GP3, GP4, M and N (van Nieuwstadt et al., J. Virol. 70,4767-4772, 1996). Interestingly, MAbs directed against GP4 were neutralizing, suggesting that at least part of the protein is exposed at the virion surface. Furthermore, most of the Mabs directed against N reacted with all PRRSV isolates tested.
PRRS in it self is a problem of major concern for the swine industry in most parts of the world. Introduction of PRRSV in pig herds will cause severe economic losses. Diagnostic testing against PRRS is widely practiced by many veterinarians and laboratories. Most diagnostic tests, such as IPMA, IFT, IFA, ELISA, each comprising suitable means of detection such as conjugated enzymes or fluorochromes, and other substrates, use interactions between antigen derived from PRRSV and antibodies directed against PRRSV to measure the presence of either PRRSV antigen or antibodies directed against PRRSV in a biological sample, such as blood, serum, tissue, tissue fluids, lavage fluids, urine, feces, that is sampled from the animal (such as a pig) to be tested. The antigen and/or antibodies used in these diagnostic tests, or diagnostic kits or assays, for PRRS diagnosis are only defined by their origin from, or by their reactivity with PRRSV. In principle this suffices for screening assays where a high specificity or sensitivity is not explicitly required. However, the ever continuing spread of PRRS has caused great concern among the pig industry, to the extent that it is deemed needed to eradicate PRRS from whole herds, or even from complete areas, regions, or countries where pigs are raised. A clear example of this need is the proposed eradication program relating to PRRS in Denmark. If one decides to completely eradicate PRRS then diagnostic tests are needed that exhibit higher specificity or sensitivity than the tests used today.
Vaccination against PRRS is also widely practiced. Several examples are known of modified live vaccines that are used, and also killed vaccines are known. However, a problem with live vaccines in general, and thus also with live PRRS vaccines, exists in that these vaccines have a tendency to spread to non-vaccinated pigs, thereby spreading instead of reducing detectable infection in pig herds, and thus being counter productive to complete eradication. If a line marker vaccine were used that could serologically be differentiated from the wild type virus, then this problem would be greatly reduced. Added disadvantages are that live vaccines sometimes cause anaphylactic reactions in the vaccinated pigs, because of undefined antigenic components. Although killed vaccines in general are reported to induce protection in the vaccinated pig, and have the additional advantage that they do not spread from pig to pig, a disadvantage of killed vaccines is that it may be hard to accrue sufficient antigenic mass in one dose of a vaccine to elicit a measurable and protective immune response. Especially killed vaccines that can induce measurable neutralizing antibody titers in pigs would be beneficial to have since measuring these neutralizing antibodies in vaccinated pig populations would help generate understanding about the level of protection obtained by vaccination in the pig herd. In addition, if one succeeds in assembling the necessary antigenic mass, this also means that more and other undefined antigenic mass is also present in the vaccine, which can also give rise to the anaphylactic reactions as described above. In this sense it would be beneficial to know which specific site on PRRSV is important peptide sequences needed for eliciting neutralizing antibodies. An advantage of the currently used vaccines originating from PRRSV isolates isolated in the U.S. is that such vaccines, albeit fully protective against and immunologically cross-reactive with European isolates of PRRSV, contain, as yet undefined, epitopes or antigenic sites by which they can be discerned from European isolates of PRRSV. Reciprocally, live vaccines originating from PRRSV isolates isolated in Europe, albeit fully protective against and immunologically cross-reactive with U.S. isolates of PRRSV, contain similar as yet undefined epitopes or antigenic sites by which they can be discerned from U.S. isolates of PRRSV.
If serological tests would be available which could discriminate (based on the small epitopic differences between PRRSV isolates) between pigs that are either vaccinated with a U.S. derived vaccine or infected with a European wild type of PRRSV (being vaccinated or not), or which could discriminate pigs that are either vaccinated with a European derived vaccine or infected with an U.S. wild type of PRRSV (being vaccinated or no), than marker vaccines and corresponding diagnostic tests (incorporating said discerning epitopes or antigenic sites) could be developed which could be used with large confidence in eradication programs for PRRS. For example, in Denmark it would than be possible to vaccinate with a U.S. derived vaccine and measure the set of antibodies in the Danish pigs which are solely directed against unique epitopes on European wild types of PRRSV and not cross-reactive with U.S. strains. This would enable the unequivocal detection and subsequent removal of wild type infected pigs from Danish herds. Currently, such a discrimination is not possible due to the overall broad immunological cross-reactivity between PRRSV isolates. It goes without saying that such combined vaccination-testing programs will be the basis for eradication of PRRS, and can also be used in other countries, if needed with distinct PRRSV antigenic sites being used in vaccine and/or diagnostic test.
The invention now provides antigenic sites comprising peptide sequences of PRRSV which allow the improvement of vaccines, be it killed or attenuated vaccines or vaccines derived via recombinant DNA technology, and antigenic sites which allow the improvement of diagnostic methods, tests and kits and the production of new diagnostic methods, tests and kits. Artificial changes or amino acid residue substitutions that maintain the antigenicity (as for example defined by the reactivity with polyclonal sera or MAbs) and thus functionality of the antigenic site can easily be derived from sequences known to constitute an antigenic site of a specific isolate by a person with ordinary skills in the art of peptide design and synthesis. For example, certain amino acid residues can conventionally be replaced by others of comparable nature, e.g. a basic residue by another basic residue, an acid by an acid, a bulky by a bulky, a hydrophobic or hydrophilic by another hydrophobic or hydrophilic residue, and so on. Also, other, less conventional but more specific changes are also possible that maintain or even improve the antigenicity of the selected sequence. Such changes can for example be made by PEPSCAN based amino acid substitutions or replacement mapping techniques (van Amerongen et. al., Peptide Research (1992) 5,269-274). In short, amino acid residues within the antigenic sites provided by the invention can e.g. be replaced conventionally or under guidance of replacement mapping, whereby the resulting peptide sequences are functionally equivalent to the antigenic site. The replacing amino acids can be either L- or D-amino acid residues. In addition, the peptide sequences provided by the invention are rendered even more immunogenic by conjugating them to adjuvants (such as KLH) known in the art. Additionally, the peptides are rendered even more immunogenic by making peptides with one (such as tandem peptides) or more repeated sequences or by polymerization or circularization.
Although it has been shown before that the N protein is immunogenic (Meulenberg (1995), J. Clin. Diagn. Lab. Immunol. 2, 652-656, GB 2 289 279 A) and that conserved and nonconserved regions between the N protein of European strains (LV) and U.S. strains (VR2332) exist (WO 96/04010), we demonstrate here for the first time which conserved and nonconserved regions are antigenic and which can be used individually or in combinations as antigens for immunization or diagnostic assays. Furthermore it is identified here that the antigenic regions in the N protein consist both of linear and conformation dependent epitopes.
The GP4 protein is the first structural protein of PRRSV for which is shown that it elicits antibodies that can neutralize the virus. A specific region of approximately 40 amino acids was identified and defined that should be exposed at the virion surface as a target for neutralizing antibodies, which then prevent the virus to infect the cells. This is an exciting new finding since it is generally assumed that the GP5 protein, the major structural of PRRSV, is the most important candidate involved in the attachment of the host cell.
The invention provides a major antigenic site, a neutralization site on GP4 of PRRSV. The invention provides the localization of a major neutralization site important for the design of effective marker vaccines that comprise amino acid core sequences and amino acid sequences flanking the core sequences of PRRSV isolates which sequences comprise the neutralization site on the ORF4 protein of PRRSV. By incorporating the relevant neutralization site sequences in the various types of vaccines, it is possible to specifically induce neutralizing antibodies in the vaccinated pig. Killed vaccines comprising the neutralization site provided by the invention are made to induce measurable neutralizing antibodies. Especially sequences located at positions in the ORF 4 encoded protein of PRRSV corresponding to those found at about amino acid 40 to 79 as found in PRRSV isolate I-1102 comprise the neutralization site. Furthermore, selected peptide sequences are made even more immunogenic by mixing the peptides with adjuvants or other carriers known in the art. The thus obtained peptide compositions are used as a vaccine. However, also the selected peptide sequences comprising the neutralization site are incorporated in vaccine vector systems, being either distinct recombinant vectors derived of heterologous viruses or bacteria, but the selected peptide sequences are also selectively incorporated in PRRSV vector viruses or vaccines derived thereof.
In a further aspect of the invention, amino acid sequences located at positions corresponding from about 52 to 75 more specifically constitute a broadly reactive neutralization site. Other embodiments of the neutralization site provided by the invention can be found among the various PRRSV isolates known or to be found (see for instance the experimental part of this description). It is easy for any person working in the field of molecular biology to compare the sequences comprising the neutralization site provided by the invention with the amino acid sequence of the ORF 4 encoded protein of yet another PRRSV isolate.
The invention also provides peptide sequences of PRRSV which improve diagnostic tests, be it antigen or antibody detection tests. The invention provides various groups of antigenic sites which are used alone or in combination in diagnostic tests. In this way diagnostic tests are provided by the invention that serve the various needs that exists in the field with regard to diagnosis and differential diagnosis. Antigen-antibody interactions always entail cross-reactive epitope-paratope interactions of amino acid sequences that are from 5 to 15 amino acid sequences long. Thus amino acid sequences of 5 to 15 amino acids long and partly or completely overlapping with the core sequences of the antigenic sites of invention are provided by the invention for incorporation in diagnostic tests. These peptide sequences are used to select or design antigen or antigenic substance containing the sequences in the test to be used. Alternatively, and provided by the invention, are synthetic antibodies reactive with the antigenic sites provided by the invention. These sites or related sequences react with synthetic antibody obtained from systems such as phage display libraries or clonal selection of (heavy chain) antibodies that constitute antibody-like molecules which can easily be expressed in heterologous expression systems.
One group provided by the invention comprises the peptide sequence corresponding to said neutralization site, as already explained above. Diagnostic tests comprising this site and/or antibodies specifically directed against this site detect neutralizing antibodies in the pig.
Another group provided by the invention comprises a conserved antigenic site on protein N. Within the conserved antigenic site the invention provides a core sequence VNQLCQLLGA (SEQ ID NO. 1) or VNQLCQMLGK (SEQ ID NO. 2). Diagnostic tests comprising this site and/or antibodies specifically directed against this site detect those antibodies in pigs that specifically react with most PRRSV isolates. Also, diagnostic tests are provided that use antibodies directed against the conserved site to detect the PRRSV antigen, thereby allowing the test to detect PRRSV isolates, irrespective of their origin.
Another group provided by the invention comprises a non-conserved differentiating antigenic site on protein N. Diagnostic tests comprising this site and/or antibodies specifically directed against this site detect those antibodies in pigs that specifically react with distinct PRRSV isolates, whereby for example vaccinated pigs can be discriminated from pigs infected with wild type PRRSV. Also, diagnostic tests are provided that use antibodies directed against the non-conserved site to detect the PRRSV antigen, thereby allowing the test to discern different PRRSV isolates. Within one such a non-conserved site the invention provides a core sequence PRGGQAKKKK (SEQ ID NO. 3) or PRGGQAKRKK (SEQ ID NO. 4) or PRGGQAKKRK (SEQ ID NO. 5) or GPGKKNKKKN (SEQ ID NO. 6) or GPGKKNKKKT (SEQ ID NO. 7) or GPGKKNRKKN (SEQ ID NO. 8) or GPGKKFKKKN (SEQ ID NO. 9) or GPGKKIKKKN (SEQ ID NO. 10) or GPGQINKKIN (SEQ ID NO. 11). Within another non-conserved site the invention provides a core sequence MAGKNQSQKK (SEQ ID NO. 12) or MPNNNGKQTE (SEQ ID NO. 13) or MPNNNGKQPK (SEQ ID NO. 14) or MPNNNGKQQK (SEQ ID NO. 15) or MPNNNGKQQN (SEQ ID NO. 16) or MPNNNGRQQK (SEQ ID NO. 17). Also, artificial changes that maintain the antigenicity and thus functionality of the above core sequences in the GP4 or N protein can easily be introduced by anyone skilled in the art of peptide design and synthesis, as described above.
The invention also provides a group comprising conformational epitopes (which vary greatly among the various isolates) which can be found at positions corresponding to those found in isolate I-1102 from amino acid position 51 to about 68 (in isolate I-1102 core sequence PKPHFPLAAEDDIRHHL) (SEQ ID NO. 18) or from 79 to about 90 (in isolate I-1102 core sequence SIQTAFNQGAGT) (SEQ ID NO. 19) or from 111 to 124 (in isolate I-1102 core sequence HTVRLIRVTSTSAS) (SEQ ID NO. 20) on protein N. The conserved and non-conserved and differentiating and conformational sites in the N protein, which sites are provided by the invention, provide diagnostic tests that unequivocally diagnose PRRSV infections. Tests are made that avoid employing non-conserved sites thereby avoiding false-negative results. In addition, the various non-conserved sites are used in the development of differentiating tests that can e.g. discriminate vaccinated pigs from pigs infected with wild type isolates of PRRSV. Again, as said it is easy for any person working in the field of molecular biology to align the sequences comprising the conserved or non-conserved or conformational epitope sites with amino acid sequences of the ORF 7 encoded protein of yet another PRRSV isolate. The sites provided by the invention are used in new pairs of vaccine-discriminating diagnostic tests for use in eradication programs of PRRS.